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United States Patent | 6,184,369 |
Rando ,   et al. | February 6, 2001 |
The oligonucleotides have sufficient guanosine to form a guanosine tetrad and can be composed of at least about 40% guanosine nucleotides, the nucleotide sequence containing at least two runs of at least two guanosines. Some of the new oligonucleotides also contain phosphorothioate backbones and 3' end modifications. Representative guanosine-rich oligonucleotides of the present invention demonstrate anti-viral activity in tissue culture against HSV-2, HIV-1, HCMV and FMLV, and show specific inhibition of bacterial RNA polymerase enzymes T7 and T3, the FMLV and HIV-1 reverse transcriptase enzyme and eukaryotic RNA polymerase.
Inventors: | Rando; Robert F. (The Woodlands, TX); Fennewald; Susan (The Woodlands, TX); Zendegui; Joseph G. (The Woodlands, TX); Oiwana; Joshua O. (Spring, TX); Hogan; Michael E. (The Woodlands, TX) |
Assignee: | Aronex Pharmaceuticals, Inc. (The Woodlands, TX) |
Appl. No.: | 535168 |
Filed: | October 23, 1995 |
PCT Filed: | April 25, 1994 |
PCT NO: | PCT/US94/04529 |
371 Date: | October 23, 1995 |
102(e) Date: | October 23, 1995 |
PCT PUB.NO.: | WO94/25037 |
PCT PUB. Date: | November 10, 1994 |
Current U.S. Class: | 536/23.1; 435/6; 536/24.5; 536/25.5 |
Intern'l Class: | C07H 021/00; C07H 021/02; C07H 021/04 |
Field of Search: | 536/23.1,24.5,25.5 514/44 |
3687808 | Aug., 1972 | Merigan et al. | 536/23. |
4388306 | Jun., 1983 | Field et al. | 424/177. |
4981957 | Jan., 1991 | LeBleu et al. | 536/25. |
5075217 | Dec., 1991 | Weber | 435/6. |
5176996 | Jan., 1993 | Hogan et al. | 435/6. |
5334711 | Aug., 1994 | Sproat et al. | 536/24. |
5397702 | Mar., 1995 | Cahalan et al. | 435/69. |
5428007 | Jun., 1995 | Fischer et al. | 514/6. |
5567604 | Oct., 1996 | Rando et al. | 435/238. |
5591721 | Jan., 1997 | Agrawal et al. | 514/44. |
Foreign Patent Documents | |||
0375408 | Jun., 1990 | EP. | |
0713705A | May., 1996 | EP. | |
8901036 | Feb., 1989 | WO. | |
WO9408053 | Apr., 1994 | WO. |
He et al., "Characterization of Human Cytomegalovirus UL84 Early Gene and Identification of Its Putative Protein Product," J. Virology, 66(2), 1098-1108 (1992). Oram et al., "Use of Recombinant Plasmids to Investigate the Structure of the Human Cytomegalovirus Genome," J. Gen. Virology, 59, 111-129 (1982). Weston et al., "Sequence of the Short Unique Region, Short Repeats, and art of theLong Repeats of Human Cytomegalovirus," J. Mol. Biology, 192, 177-208 (1986). Tamashiro et al.(I), "Structure of the Heterogeneous L-S Junction Region of Human Cytomegalovirus Strain AD169 DNA," J. Virology, 52(2), 541-548 (1984). Mocarski et al., "Structure and Variability of the a Sequence in the Genome of Human Cytomegalovirus (Towne Strain)," J. Gen.Virology, 68, 2223-2230 (1987). Tamashiro et al.(II), "Terminal Structure and Heterogeneity in Human Cytomegalovirus Strain AD 169," J. Virology, 59(3), 591-604 (1986). Hennighausen et al., "Nuclear Factor 1 Interacts with Five DNA Elements int eh Promoter Region of the Human Cytomegalovirus Major Immediate Early Gene," EMBO J., 5(6), 1367-1371 (1986). Rasmussen et al., "Sequences in Human Cytomegalovirus Which Hybridize with the Avian Retrovirus Oncongene v-myc Are G+C Rich and Do Not Hybridize with the Human c-myc Gene," Molecular & Cellular Biology, 5(6), 1525-1530 (1985). G. Zon, "Oligonucleotide Analogues as Potential Chemotherapeutic Agents," Pharmaceutical Research, 5(9), 539-549 (1988). Miller et al., "Control of Ribonucleic Acid Function by Oligonucleoside Methylphosphonates," Biochemie, 67, 769-776 (1985). Marshall et al., "Phosphorodithioate DNA as a Potential Therapeutic Drug," Science, 259, 1564-1570 (1993). Gura, "Antisense Has Growing Pains--Efforts to Develop Antisense Compounds for Cancer, AIDS, and Other Diseases Have Encountered Some Unexpected Questions About How the Drugs Really Work," Science, 270, 575-577 (1995). Kreig et al., "CpG Motifs in Bacterial DNA Trigger Direct B-Cell Activation," Nature, 374, 546-549 (Apr. 6, 1995). Patrick et al., "Antiviral and Resistance Studies of AG1343, an Orally Bioavailable Inhibitor of Human Immunodeficiency Virus Protease," Antimicrobial Agents and Chemotherapy, 40(2), 292-297 (Feb., 1996); supplied but not cited by applicant. Rusconi et al., "Naphthalene Sulfonate Polymers with CD4-Blocking and Anti-Human Immunodefiency Virus Type 1 Activities," Antimicrobial Agents and Chemotherapy, 40(1), 234-236 (Jan. 1996); supplied but not cited by applicant. Wallace et al.(I), "Pharmacokinetics and Distribution of a .sup.33 P-Labeled Anti-Human Immunodeficiency Virus Oligonucleotide (AR177) After Single- and Multiple-Dose Intravenous Administration to Rats," J. Pharmacology and Experimental Therapeutics, 280(3), 1480-1488 (1997); supplied but not cited by applicant. Wallace et al. (II), "Single-Dose Hemodynamic Toxicity and Pharmacokinetics of a Partial Phosphorothioate Anti-HIV Oligonucleotide (AR177) After Intravenous Administration to Cynomolgus Monkeys," J. Pharmacology and Experimental Therapeutics, 278(3), 1306-1312 (1996); supplied but not cited by applicant. Wallace et al. (III), "Repeat-Dose Toxicity and Pharmacokinetics of a Partial Phosphorothioate Anti-HIV Oligonucleotide (AR177) After Bolus Intravenous Administration to Cynomolgus Monkeys," J. Pharmacology and Experimental Therapeutics, 278(3), 1313-1317 (1987); supplied but not cited by applicant. Rando, "Clinical Trial Results of Aronex's Anti-HIV Oligonucleotide (AR177) and Recent Antisense Technology Advances," IBC's Fourth International Symposium on Antisense Therapeutics with New Applications for Genomics, International Business Communications, Inc., Wyndham Emerald Plaza Hotel, San Diego, CA, Feb. 6-7, 1997; only abstract supplied; supplied but not cited by applicant. Clinical Update, Hybridon, Inc., Worcester, MA, Feb. 10, 1997; press release apparently obtained from the Internet; supplied but not cited by applicant. Hybridon Moves GEM.RTM. 91 into Confirmatory Clinical Trial in Advanced HIV-Positive Patient, Hybridon, Inc., Cambridge, MA, Feb. 10, 1997; original release date was Feb. 6-7, 1997 in San Diego, CA (See ref. RB supra); supplied but not cited by applicant. Kahn et al., "Phase 1 Study of AR-177 (Zintevir), an HIV-1 Inhibitor with Significant Activity Against Integrase Protein: Safety, Pharmacokinetics, Immunologic and Virologic Activity," Abstract of presentation at the 11th International Conference on AIDS, Vancouver, BC, Jul. 7-12, 1996; supplied but not cited by applicant. Kern, "Preclinical Evaluation of Antiviral Agents: In Vitro and Animal Model Testing," Ch. 3 in Antiviral Agents and Viral Diseases in Man, Galasso et al. (eds.), Raven Press, Ltd., New York, NY, 1990, pp. 87-114, only pp. 87 and 94-95 supplied. Balzarini, J., "Suppression of the Breakthrough of Human Immunodeficiency Virus Type 1 (HIV-1) in Cell Culture by Thiocarboxanilide Derivatives When Used Individually or in Combination with Other HIV-1-Specific Inhibitors (i.e., TSAO Derivatives)," Proc. Natl. Acad. Sci. USA 92:5470-5474 (Jun. 1995). Nagy, K. et al., "Antiviral Activity of Human Immunodeficiency Virus Type 1 Protease Inhibitors in a Single Cycle of Infection: Evidence for a Role of Protease in the Early Phase," J. Virol. 68:757-765 (Feb. 1994). Nelson el al., "Bifunctional Oligonucleotide Probes Synthesized Using a Novel CPG Support Are Able to Detect Single Base Pair Mutations," Nucleic Acids Research, 17:7187-7194 (1989). (issue No. 18). Nelson et al., "A New and Versatile Reagent for Incorporating Multiple Primary Aliphatic Amines into Synthetic Oligonucleotides," Nucleic Acids Research, 17: 7179-7186 (1989). (issue No. 18). Vlassov et al, "The Effect of Modification of Terminal Groups of Oligonucleotides on Their Stability in Mycoplasma Culture," Biopolim. Kletka, vol. 7, No. 5, (Novosibirsk, USSR), pp. 37-41, see Biosis, Abstract No. 94-032,483, (1994). Zendegui et al, "In Vivo Stability and Kinetics of Absorption and Disposition of 3'Phosphopropyl Amine Oligonucleotides," Nucleic Acids Research, 20: 307-314 (1992). (issue No. 2). Agrawal S. et al., "Oligodeoxynucleoside Phosphoramidates and Phosphorothioates as Inhibitors of Human Immunodeficiency Virus" Proceedings of the National Academy of Sciences of USA, vol. 85, Oct. 1, 1998, pp. 7079-7083. Wyatt, J. et al., "Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion" Proceedings of the National Academy of Sciences of USA., vol. 91, Feb. 1994. Rando, R. et al., "Suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads" Journal of Biological Chemistry., vol. 270, Jan. 27, 1995, pp. 1754-1760. Supplementary Partial European Search Report, dated Jul. 16, 1998, that was received in EPC counterpart application EP 94 917899, filed on Apr. 25, 1994. |
TABLE 1 SEQ ID NO 5(B106-62) 5'-gtggtggtggtgttggtggtggtttggggggtgggg-3' SEQ ID NO 6(B106-71) 5'-gtggttggtggtggtgtgtgggtttggggtgggggg-3' SEQ ID NO 21(I100-01) 5'-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-3' SEQ ID NO 24(I100-07) 5'-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3' SEQ ID NO 28(I100-50) 5'-ggtggtggggtggttgttgggggttg-3' SEQ ID NO 29(I100-51) 5'-ggtggtggggtggttgttgggggttgttgggggtgtgtgggtggt-3' SEQ ID NO 26(I100-11) 5'-gatccatgtcagtgacactgcgtagatccgatgatccagtcgatg-3' SEQ ID NO 12(G101-50) 5'-ggtgggtggtttgtgtggttggtgggtttt-3' SEQ ID NO 13(G105-50) 5'-ggggggggggtgtgggggggggttgtggtgg-3' SEQ ID NO 14(G106-50) 5'-ggtgggtgggttggggggtgggtgggg-3' SEQ ID NO 15(G109-50) 5'-tggggtttgggtggggggttgggtggttg-3' SEQ ID NO 16(G110-50) 5'-gggtggtggtgttggtgttgtgtg-3' SEQ ID NO 17(G113-50) 5'-ggtgggggggttggtgtgtttg-3' SEQ ID NO 1(A100-00) 5'-tgggtggggtggggtgggggggtgtggggtgtggggtg'3' SEQ ID NO 2(A100-50) 3'-tgggtggggtggggtgggggggtgtggggtgtggggtg-5' SEQ ID NO 4(A101-00) 5'-ggtggtgggggggggtggggtggtggtgggggtgttgg-3' SEQ ID NO 18(HIV26ap) 5'-gtgtgggggggtggggtggggtgggt-3' SEQ ID NO 19(HIV26ctl) 5'-gggtgggtgggtgggtgggtgggtgg-3' SEQ ID NO 9(B107-51) 5'-ggtggggtggtggtggttggggggggggggt-3' SEQ ID NO 10(B133-55) 5'-ggtggttggggggtggggggg-3' SEQ ID NO 11(B133-55) 5'-gggtggggtggtgggtggggg-3' SEQ ID NO 20(I100-00) 5'-gttgggggttgttggtggggtggtgg-3' SEQ ID NO 27(I100-12, PT) 5'-gttgggggttgttggtggggtggtgg-3' SEQ ID NO 22(I100-05) 5'-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg- CHOL SEQ ID NO 23(I100-06) 5'-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg- CHOL SEQ ID NO 25(I100-08) 5'-gttgggggttgttggtggggtggtgg-CHOL SEQ ID NO 3 5'-gggtgggtgggtgggtgg-3' SEQ ID NO 30 5'-gggtggttgggtggttgg-3' SEQ ID NO 31(1173) 5'-gggtgggtgggtgggtgg-3' SEQ ID NO 32(1174, PT) 5'-gggtgggtgggtgggtgg-3' SEQ ID NO 33(I100-15) 5'-gtggtgggtgggtgggt-3' SEQ ID NO 34(I100-16) 5'-gtggtgggtgggtgggtggtgggtggt-3' SEQ ID NO 35(I100-17) 5'-gtggtgggtgggtgggtggtgggtggtggttgtgggt-3' SEQ ID NO 36(I100-18) 5'-ttgtgggtgggtggtg-3' SEQ ID NO 37(I100-19) 5'-tggtgggtggtggttgtgggtgggtggtg-3' SEQ ID NO 38(I100-20) 5'-gtgggtgggtggtgggtggtggttgtgggtgggtggtg-3' SEQ ID NO 39(I100-21, PT) 5'-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3' SEQ ID NO 40(1231) 5'-gatccatgtcagtgacac-3' SEQ ID NO 41(1232, PT) 5'-gatccatgtcagtgacac-3' SEQ ID NO 42(1229) 5'-cccccccccccccccccc-3' SEQ ID NO 43(1230, PT) 5'-cccccccccccccccccc-3' SEQ ID NO 44(1198) 5'-ttcatttgggaaacccttggaacctgactgactggccgtcgttttac-3' SEQ ID NO 45(1200) 5'-gtaaaacgacggcca-3' SEQ ID NO 46(I100-25) 5'-gtggtgggtgggtgggg-3' SEQ ID NO 47(I100-26) 5'-gtggtgggtgggtggg-3' SEQ ID NO 48(I100-35) 5'-tggtgggtgggtgggt-3' SEQ ID NO 49(I100-27) 5'-gtggtgggtgggt-3' SEQ ID NO 50(I100-28) 5'-gtggtgggt-3' SEQ ID NO 51(I100-30) 5'-gtgggtgggtgggt-3' SEQ ID NO 52(I100-29) 5'-gtgggtgggt-3'
TABLE 2 ED.sub.50 for oligonucleotides in an anti-HIV-1 syncytia formation assay. G-Rich oligonucleotide ED50 I100-00 3.75 .mu.M I100-01 4.50 .mu.M I100-05 3.25 .mu.M I100-08 3.25 .mu.M I100-06 0.70 .mu.M I100-07 0.25 .mu.M A100-00 3.25 .mu.M
TABLE 3 Guanosine/thymidibne and control oligonucleotide sequences Oligo.sup.a Length 3'-Modification.sup.b Sequence TC.sub.50.sup.c I100-07 45 mer amine 5'-gtggtggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3' >50 .mu.M I100-06 45 mer cholestreol 5'-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3' I100-00 26 mer amine 5'- gttgggggttgttggtggggtggtgg-3' 37 .mu.M I100-08 26 mer cholestrol 5'- gttgggggttgttggtggggtggtgg-3' I100-12 26 mer amine(PT) 5'- gttgggggttgttggtggggtggtgg-3' 18 .mu.M I100-01 45 mer amine 5'-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-3' I100-05 45 mer cholestrol 5'-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-3' A100-00 38 mer amine 5'-tgggtggggtggggtgggggggtgtggggtgtggggtg -3' 1173 18 mer amine 5'-gggtgggtgggtgggtgg -3' I100-11 45 mer amine 5'- gatccatgtacgtgacactgcgtagtccgatgatcagtcgatg-3' 46.5 .mu.M 1231 18 mer amine 5'-gatccatgtcagtgacac -3' 1229 18 mer amine 5'-cccccccccccccccccc -3' .sup.a All oligonnucleotides listed were synthesized with phosphodiester backbones except I100-12 which had phosphorothioate (PT) linkages. .sup.b The capping group at the 3'-end of the oligonucleotide was either a propanolamine or cholestrol moiety. .sup.c Median inhibitory (toxic) concentration in tissue culture.
TABLE 4 Guanosine/thymidine oligonucleotide sequences. ED50.sup.b (uM) Oligo Length linkage.sup.a Sequence Syn p24 T.I..sup.c I100 -07 45 mer PD 5'- gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg 0.25 0.55 -27 45 mer PT 5'- gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg 0.225 <0.20 >100 -20 38 mer PD 5'- gtgggtgggtggtgggtggtggttgtgggtgggtggtg 1.00 1.00 -19 29 mer PD 5'- tggtgggtggtggttgtgggtgggtggtg 3.75 2.00 -18 16 mer PD 5'- ttgtgggtgggtggtg 3.75 3.00 -17 37 mer PD 5'- gtggtgggtgggtgggtggtgggtggtggttgtgggt 0.30 0.20 -16 27 mer PD 5'-gtggtgggtgggtgggtggtgggtggt 0.25 0.15 >200 -15 17 mer PD 5'-gtggtgggtgggtgggt 0.125 0.08 >200 -00 26 mer PD 5'-gttgggggttgttggtggggtggtgg 3.25 ND -12 26 mer PT 5'-gttgggggttgttggtggggtggtgg 0.225 <0.20 AZT 0.04 0.40 >200 .sup.a The internucleotide backbone linkages are denoted as PD for phosphodiester and PT for phosphorothioate. .sup.b The ED50 values for the syncytium and p24 inhibition assays in uM concentrations. .sup.c T.I. = therapeutic index.
TABLE 5 Inhibition of HIV-1 Induced Syncytia Using Size variants of 1100-15. oligo Sequence ED50 Syn. (uM) I100-15* 5' gtggtgggtgggtgggt -3' 0.16 I100-25 5' gtggtgggtgggtgggg -3' 0.25 I100-26* 5' gtggtgggtgggtggg -3' 0.12 I100-35 5' tggtgggtgggtgggt -3' 1.75 I100-27 5' gtggtgggtgggt -3' 4.50 I100-28 5' gtggtgggt -3' 4.50 I100-30 5' gtgggtgggtgggt -3' 4.50 I100-29 5' gtgggtgggt -3' >10.00 AZT 0.02 *At 5 uM these compounds suppressed virus at least 7 days post-removal of drug. All other compounds at 5 uM wre the same as AZT 7 days after removal of drug.
TABLE 6 Detection of HIV-1 p24 antigen in the culture media of GTO treated SUP T1 cells. Percent p24.sup.a Oligonucleotide (2.5 uM) Day 4.sup.b Day 7 Day 11 Control SUP T1 cells 100.0% 100.0% 100.0% I100-07 6.0% 15.9% 8.6% I100-21 (PT).sup.d 0.0% 0.0% 0.0% I100-15 0.0% 0.0% 0.0% I100-16 0.0% 0.0% 0.0% I100-18 144.5% 9.7% 5.3% I100-19 208.0% 21.8% 15.0% I100-12 (PT) 0.0% 0.0% 0.0% .sup.a Level of detectable p24 in culture medium relative to control (infected but untreated SUP T1 cells after subtraction of background values. .sup.b Day 4 post-infection culture medium was replaced with fresh medium without oligonucleotide. .sup.c SUP T1 cells infected with HIV-1 but not treated with oligonucleotides or AZT were used as positive control cells in this experiment. .sup.d 1100-21 and 1100-12 contain phosphorothioate backbone linkages (PT).
TABLE 7 In Vitro Inhibition of HIV-1 RT by PD and PT Oligonucleotides. Oligonucleotides Length Linkage.sup.b Ki (.mu.M) ID50 (.mu.M) I 100-00 26 PD 0.37 5.0 I 100-12 26 PT 0.005 0.015 I 100-07 45 PD 0.137 2.5 I 100-21 45 PT 0.001 0.004 I 100-15 17 PD >5.0 >5.0 1173 18 PD >5.0 >5.0 1174 18 PT 0.015 0.0154 1229 (poly dC) 18 PD >5.0 >5.0 1230 (poly dC) 18 PT 0.044 0.033 1231 (GATC) 18 PD >5.0 >5.0 1232 (GATC) 18 PT 0.56 0.045 .sup.a Each pair of oligonucleotides contain the same sequence and differ only in the nature of their backbone linkage. Oligonucleotides 1229 and 1230 were poly dC while the 1231 and 1232 oligonucleotides were a random sequence of all four bases (GATC). .sup.b The backbone modifications are denoted as PD for phosphodiester and PT for phosphorothioate.
TABLE 8 In Vitro Inhibition of HIV-1 gp120 Interaction with CD4 by PD and PT Oligonucleotides. Oligonucleotide Linkage.sup.a ID50 [gp120](.mu.M) ID50[CD4](.mu.M) I 100-00 PD 3.50 18 I 100-12 PT 0.08 0.475 I 100-07 PD 0.80 4.25 I 100-21 PT 0.07 0.048 1173 PD >100 >100 1174 PT 0.09 0.45 1229 (poly dC) PD >100 >100 1230 (poly dC) PT 1.00 3.25 1231 (GATC) PD >100 >50 1232 (GATC) PT >10 >10 .sup.a Each pair of oligonucleotides contain the same sequence and differ only in the nature of their backbone linkage. .sup.b The backbone modifications are denoted as PD for phosphodiester and PT for phosphorothioate.
TABLE 9 Oligonucleotide Inhibition of HCMV Activity Viral Yield in plaque forming units (PFU) oligonucleotide (% G) G101-50 (53%) G105-50 (80%) G106-50 (78%) G109-50 (65%) G113-50 (64%) [oligo] 30 mer 31 mer 27 mer 29 mer 24 mer None 4.5 .times. 10.sup.3 PFU 4.5 .times. 10.sup.3 PFU 4.5 .times. 10.sup.3 PFU 4.5 .times. 10.sup.3 PFU 4.5 .times. 10.sup.3 PFU 20.0 .mu.M .O slashed. 4.5 .times. 10.sup.1 PFU 2.5 .times. 10.sup.1 PFU 8.0 .times. 10.sup.1 PFU 3.5 .times. 10.sup.1 PFU 10.0 .mu.M 2.5 .times. 10.sup.1 PFU 1.8 .times. 10.sup.2 PFU 4.0 .times. 10.sup.1 PFU 4.5 .times. 10.sup.1 PFU 4.0 .times. 10.sup.1 PFU 1.0 .mu.M 7.0 .times. 10.sup.2 PFU 1.9 .times. 10.sup.2 PFU 6.0 .times. 10.sup.1 PFU 1.5 .times. 10.sup.2 PFU 5.0 .times. 10.sup.2 PFU 0.5 .mu.M 8.0 .times. 10.sup.2 PFU 2.7 .times. 10.sup.2 PFU 1.3 .times. 10.sup.2 PFU 3.0 .times. 10.sup.2 PFU 5.4 .times. 10.sup.2- PFU